Compositions comprising folic acid derivatives, their preparations and methods of use

ABSTRACT

Described herein are methods for making folic acid derivatives, intermediates, pharmaceutical compositions and uses thereof.

CROSS-REFERENCE AND CLAIM OF PRIORITY

This application is a divisional of U.S. Ser. No. 15/912,070, filed Mar.5, 2018, which is a continuation of U.S. Ser. No. 15/173,164, filed Jun.3, 2016, which is a continuation of U.S. Ser. No. 14/417,504, filed Jul.26, 2013, which is a National Stage Application under 35 U.S.C. § 371 ofInternational Application No. PCT/US2013/052304, filed Jul. 26, 2013,which claims priority to U.S. Ser. No. 61/676,651, filed Jul. 27, 2012,the contents of each of which is hereby incorporated by reference in itsentirety.

BACKGROUND OF INVENTION

Folic acid (FA), also known as pteroyl-L-glutamic acid, is a vitalco-factor in enzymatic reactions necessary for the synthesis of nucleicacids, amino acids, and other biological molecules. Although manyorganisms are capable of synthesizing folic acid, humans are unable tosynthesize folic acid, and must depend on adequate dietary intake ofthis essential nutrient.

Without adequate intake of folic acid, humans may develop a folatedeficiency. Folate deficiency has several negative impacts on the humanbody, including but not limited to: (i) the defective maturation ofdifferent cell types; (ii) nervous system disorders; (iii) a decreasedimmune response; and (iv) the development of peripheral vasculardisease. It has also been found that insufficient folate levels duringpregnancy correlate with the occurrence of neural tube defects innewborns. Low folate levels may also lead to megaloblastic anemia, adisorder which results in inadequate production of red blood cells,particularly during pregnancy and in geriatrics.

The clear connection between adequate folate intake and health hasresulted in the establishment of a recommended dietary allowance (RDA)for folic acid by the U.S. government. Although folic acid is currentlyadded to all commercial over the counter (OTC) vitamin preparations, andto some foods, folic acid is not the primary form of folate which isfound naturally in fresh foods. More commonly, the primary forms offolate which are found in natural fresh foods are polyglutamates. Ofthese polyglutamates, the polyglutamate forms of (6S)-methylTHFA (e.g.,the compound of formula (I) where R¹ is NH₂, R² is OH and R³ is H) and(6S)-5-formylTHFA predominate. However, since the primary form of folatewhich can be absorbed by the human body bears only a single glutamicacid residue, polyglutamates, after ingestion, must be processedenzymatically in the digestive tract prior to absorption. Anotherdifference between folic acid and natural folates (e.g. polyglutamates)is that the folates in fresh, uncooked foods are usually present as areduced form. One example of a reduced folate is tetrahydrofolic acid(THFA) and its derivatives.

There is a reason to believe that in a segment of the population theabsorption of reduced folates, such as tetrahydrofolic acid (THFA), mayexceed folic acid, resulting in greater bioavailability. Thus, dietary,supplementation with these reduced folates (e.g., THFA) may constitutean improved method for meeting the RDA of folic acid. In fact, thecalcium salt of (6S)-methylfolate (Formula (I); X=Ca), also known as,L-methylfolate, is currently commercially available under the trade nameMetafolin™ for use as a dietary supplement. In addition to theforegoing, (6S)-5-methylfolate may be the body's preferred form offolate, since it is the predominant form of folate found in humans.

Although the importance of folic acid in the diet has been recognized,prior and widespread use of reduced folates as dietary supplements hasbeen limited, in part by the stereochemistry of these compounds. Thechemical structure of folic acid contains a single chiral center in theglutamic acid portion of the molecules (see formula (I), where thechiral center is denoted by an asterisk). Reduction of folic acid toTHFA creates a second chiral center at the 6-position of the pteridinenucleus. When the reduction of folic acid is carried out chemically, amixture of two isomers called diastereomers (or more appropriately,epimers) results, whereby the orientation of substituents at the6-position in each isomer is different. Each of these distinctorientations, or configurations, is designated as either S or R inaccordance with the Cahn-Ingold-Prelog convention. As a result of theaforementioned reduction of folic acid, one-half of the molecules havethe S-configuration at the 6-position, and one-half of the moleculeshave the R-configuration at the 6-position. Conversely, reduction offolic acid enzymatically, e.g., by the enzyme dihydrofolate reductase(DHFR), proceeds stereoselectively, and results in only the productionof (6S)-THFA. It is important to note that all tetrahydrofolates thatoccur naturally are found in only one diasterereomeric form, i.e., theabsolute configuration at the 6-position is either S or R. Accordingly,the S designation is assigned to (i) naturally-occurring THFA, (ii)5-methylTHFA, and (iii) 5-formylTHFA, whereas the R designation isassigned to (i) naturally-occurring 5,10-methyleneTHFA, and (ii)10-formylTHFA.

In addition to the naturally occurring diastereomeric forms of reducedfolates, it has been found that some unnatural isomers of reducedfolates (i.e., those in which the configuration at the 6-position isopposite that of natural isomers) can exhibit considerable absorption inthe human gastrointestinal (GI) tract. However, a low order ofbiological activity has been ascribed to the unnatural isomers and, moreimportantly, it appears that the unnatural isomers may have aninhibitory effect upon certain enzymatic processes. Due to thesefactors, and because the effect of chronic or long-term exposure tothese unnatural isomers is unknown, there has been a recent trend to useonly diastereomerically pure (or natural) (6S)-isomers of reducedfolates as therapeutic agents, e.g., (6S)-5-formylTHFA or calciumleucovorin, and dietary supplements (e.g., Metafolin™).

A variety of methods are currently available for the production of thesedesirable pure folate isomers. At present, the methods which are usedfor commercial production of folate isomers rely on the resolution ofpairs of diastereomers, particularly by fractionalcrystallization/recrystallization techniques. For example, Metafolin™ isproduced by such a method. Some of these methods produce large volumesof undesirable by-products which need to be removed, and thus negativelyinfluence the economy and efficiency of the process. Others of thesemethods require multiple fractionations/recrystallizations to achieve aproduct of high diastereomeric excess, and therefore can betime-consuming and costly.

In addition to the foregoing, there is an approach which uses thechromatographic separation of diastereomers, but it does not lend itselfto large-scale production of pure folate isomers. Furthermore, there isa method which synthesizes (6S)-THFA via the stereoselective catalytichydrogenation of dihydrofolic acid (DHFA), but the cost of the exoticorganometallic catalyst(s) is prohibitive for large-scale production. Achemoenzymatic method has also been described for producing smallquantities of (6S)-THFA and derivatives, but this latter method istypically regarded as unsuitable for commercial application due to itscomplexity (Tetrahedron 1986, 42, 117-136).

Thus, a need remains for providing a cost-effective, large-scalesynthesis for producing L-methylfolate.

SUMMARY OF INVENTION

Disclosed herein are compositions (e.g., oral dosage forms and vitaminsupplements) of a compound of formula (V), methods of making a compoundof formula (V) and methods of making a compound of formula (I).

In one aspect, the present invention is directed to a composition (e.g.,a pharmaceutical composition) of a compound of formula (V):

wherein,R¹ and R² are each independently selected from H, OH, NH₂, C₁₋₆ alkyl oraryl;R³ is H, C₁₋₆ alkyl or aryl; andY is halo.

In some embodiments, R¹ is NH₂. In some embodiments, R² is OH. In someembodiments, R³ is H. In some embodiments, R¹ is NH₂, R² is OH and R³ isH.

In some embodiments, Y is chloro.

In another aspect, the present invention is directed to an oral dosageform comprising a compound of formula (V).

In another aspect, the present invention is directed to a dietarysupplement comprising a compound of formula (V). In some embodiments,the dietary supplement is a prenatal vitamin comprising a compound offormula (V). In embodiments, when a composition or oral dosage formcomprising a compound of formula (V) is administered to a subject, thecompound of formula (V) converts in vivo to a bioavailable form offolate.

In some embodiments, the dietary supplement further comprises one ormore vitamins (e.g., vitamin A, C, B-12 or D).

In some embodiments, the composition, oral dosage form or dietarysupplement is substantially free of any compounds of formula (I), (II)or (III):

wherein each of R¹, R², R³, and X are defined as in formula (V). In someembodiments, the composition of formula (V) is substantially free ofsolvent, e.g., water or ethanol.

In another aspect, the present invention is directed to a method formaking a compound of formula (V), the method comprising converting acompound of formula (II) into a compound of formula (V), for example, bycyclization of the compound of formula (II) to form a compound offormula (V):

wherein,R¹ and R² are each independently selected from H, OH, NH₂, C₁₋₆ alkyl oraryl;R³ is H, C₁₋₆ alkyl or aryl; andX is Na, K, Mg or Ca.

In certain embodiments, the cyclization step is carried out in thepresence of an acid such as formic acid. In some embodiments, thecyclization step is carried out in the presence of a strong acid (e.g.,an aqueous HCl solution). In some embodiments, the cyclization iscarried out in the presence of a mixture of formic and hydrochloricacid. In some embodiments, the cyclization step comprises a solvent(e.g., an aqueous solvent). The reaction can be performed under ambientconditions, for example, room temperature and atmospheric pressure.

In some embodiments, the method comprises isolation of the compound offormula (V), for example, by filtration.

In some embodiments, the compound of formula (V) is further converted toa compound of formula (I)

The compound of formula (V) can be converted into a compound of formula(I) by subjecting the compound of formula (V) to a reducing agent, forexample, a hydride such as NaBH₄. In some embodiments, the reducingagent is in a basic solution such as a NaOH solution. In someembodiments, the method further comprises subjecting the reactionmixture of a sale such as a Ca salt (e.g., CaCl₂).

In certain embodiments, R¹ is NH₂. In some embodiments, R² is OH. Insome embodiments, R³ is H. In some embodiments, R¹ is NH₂, R² is OH andR³ is H. In some embodiments, R¹ is NH₂, R² is OH and R³ is H.

In some embodiments, X is Ca.

In another aspect, the invention is directed to a method for making acompound of formula (I):

the method comprising a reduction of a compound of formula (V) asdefined above, to make a compound of formula (I),

whereinR¹ and R² are each independently selected from H, OH, NH₂, C₁₋₆ alkyl oraryl;R³ is H, C₁₋₆ alkyl or aryl;Y is halo; andX is Na, K, Mg or Ca.

In certain embodiments, the reduction of a compound of formula (V) iscarried out in the presence of a reducing agent (e.g., sodiumborohydride). In some embodiments, the reduction step further comprisesa strong base (e.g., sodium hydroxide). In some embodiments, thereduction step is carried out in the presence of a solvent (e.g., anaqueous solvent).

In certain embodiments, R¹ is NH₂. In some embodiments, R² is OH. Insome embodiments, R³ is H. In some embodiments, R¹ is NH₂, R² is OH andR³ is H. In some embodiments, X is Ca.

In some embodiments, the method further comprises converting a compoundof formula (II) to a compound of formula (V), for example, using amethod described above.

In certain embodiments, the methods described herein are carried out onat least 50 g of the starting material. In some embodiments, the methodsdescribed herein are carried out on at least 100 g of the startingmaterial. In some embodiments, the methods described herein are carriedout on at least 250 g of the starting material. In some embodiments, themethods described herein are carried out on at least 400 g of thestarting material. In some embodiments, the methods described herein arecarried out on at least 500 g of the starting material. In someembodiments, the methods described herein are carried out on at least 1kg of the starting material. In some embodiments, a method describedherein is performed as a batch process.

Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5^(th) Edition, John Wiley & Sons, Inc., New York, 2001;Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., NewYork, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd)Edition, Cambridge University Press, Cambridge, 1987; the entirecontents of each of which are incorporated herein by reference.

Certain compounds of the present invention can comprise one or moreasymmetric centers, and thus can exist in various isomeric forms, e.g.,stereoisomers and/or diastereomers. Thus, compounds and pharmaceuticalcompositions thereof may be in the form of an individual enantiomer,diastereomer or geometric isomer, or may be in the form of a mixture ofstereoisomers. In certain embodiments, the compounds of the inventionare enantiopure compounds. In certain embodiments, mixtures ofstereoisomers or diastereomers are provided.

Where a particular enantiomer or diastereomer is preferred, it may, insome embodiments be provided substantially free of the correspondingenantiomer and/or diastereomers, and may also be referred to as“optically enriched.” “Optically-enriched,” as used herein, means thatthe compound is made up of a significantly greater proportion of oneenantiomer or diastereomer. In certain embodiments the compound is madeup of at least about 90% by weight of a preferred enantiomer ordiastereomer. In other embodiments the compound is made up of at leastabout 95%, 98%, or 99% by weight of a preferred enantiomer ordiastereomer. Preferred enantiomers or diastereomers may be isolatedfrom racemic mixtures by any method known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts or prepared by asymmetricsyntheses. See, for example, Jacques, et al., Enantiomers, Racemates andResolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al.,Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of CarbonCompounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables of ResolvingAgents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of NotreDame Press, Notre Dame, Ind. 1972).

The compounds of this invention may contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures.Described herein are enantiomerically enriched compounds (e.g., acompound resolved to an enantiomeric excess of 60%, 70%, 80%, 85%, 90%,95%, 99% or greater). All such isomeric forms of these compounds areexpressly included in the present invention. The compounds of thisinvention may also contain linkages (e.g., carbon-carbon bonds) orsubstituents that can restrict bond rotation, e.g. restriction resultingfrom the presence of a ring or double bond. Accordingly, all cis/transand E/Z isomers are expressly included in the present invention. Thecompounds of this invention may also be represented in multipletautomeric forms, in such instances, the invention expressly includesall tautomeric forms of the compounds described herein, even though onlya single tautomeric form may be represented (e.g., alkylation of a ringsystem may result in alkylation at multiple sites, the inventionexpressly includes all such reaction products). All such isomeric formsof such compounds are expressly included in the present invention. Allcrystal forms of the compounds described herein are expressly includedin the present invention.

Methods for separating a racemic mixture of two enantiomers includechromatography using a chiral stationary phase (see, e.g., “ChiralLiquid Chromatography,” W. J. Lough, Ed. Chapman and Hall, New York(1989)). Enantiomers can also be separated by classical resolutiontechniques. For example, formation of diastereomeric salts andfractional crystallization can be used to separate enantiomers. For theseparation of enantiomers of carboxylic acids, the diastereomeric saltscan be formed by addition of enantiomerically pure chiral bases such asbrucine, quinine, ephedrine, strychnine, and the like. Alternatively,diastereomeric esters can be formed with enantiomerically pure chiralalcohols such as menthol, followed by separation of the diastereomericesters and hydrolysis to yield the free, enantiomerically enrichedcarboxylic acid. For separation of the optical isomers of aminocompounds, addition of chiral carboxylic or sulfonic acids, such ascamphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid canresult in formation of the diastereomeric salts. For example a compoundcan be resolved to an enantiomeric excess (e.g., 60%, 70%, 80%, 85%,90%, 95%, 99% or greater) via formation of diasteromeric salts, e.g.with a chiral base, e.g., (+) or (−) α-methylbenzylamine, or via highperformance liquid chromatography using a chiral column. In someembodiments a product is purified directly on a chiral column to provideenantiomerically enriched compound.

The “enantiomeric excess” or “% enantiomeric excess” of a compositioncan be calculated using the equation shown below. In the example shownbelow a composition contains 90% of one enantiomer, e.g., the Senantiomer, and 10% of the other enantiomer, i.e., the R enantiomer.ee=(90−10)/100=80%. Thus, a composition containing 90% of one enantiomerand 10% of the other enantiomer is said to have an enantiomeric excessof 80%.

The term “halo” or “halogen” refers to any radical of fluorine,chlorine, bromine or iodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers toan alkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). The terms “arylalkyl” or “aralkyl” refer toan alkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “arylalkyl” or “aralkyl”include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl,and trityl groups.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom capable of substitutioncan be substituted (e.g., by one or more substituents). Examples of arylmoieties include, but are not limited to, phenyl, naphthyl, andanthracenyl.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Anyatom can be substituted. Suitable substituents include, withoutlimitation, alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11,C12 straight or branched chain alkyl), cycloalkyl, haloalkyl (e.g.,perfluoroalkyl such as CF₃), aryl, heteroaryl, aralkyl, heteroaralkyl,heterocyclyl, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl,alkoxy, haloalkoxy (e.g., perfluoroalkoxy such as OCF₃), halo, hydroxy,carboxy, carboxylate, cyano, nitro, amino, alkyl amino, SO₃H, sulfate,phosphate, methylenedioxy (—O—CH₂—O—), ethylenedioxy, oxo, thioxo (e.g.,C═S), imino (alkyl, aryl, aralkyl), S(O)_(n)alkyl (where n is 0-2),S(O)_(n) aryl (where n is 0-2), S(O)_(n) heteroaryl (where n is 0-2),S(O)_(n) heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl,cycloalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, and combinationsthereof), ester (alkyl, aralkyl, heteroaralkyl, aryl, heteroaryl), amide(mono-, di-, alkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, andcombinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl,heteroaralkyl, and combinations thereof). In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents. In another aspect, a substituent mayitself be substituted with any one of the above substituents.

DETAILED DESCRIPTION

Compositions and Oral Dosage Forms

The present invention also features compositions such as pharmaceuticalcompositions, dietary supplements, and oral dosage forms of a compoundof formula (V), either alone or in combination, together with a suitableexcipient. In some preferred embodiments, the composition (e.g.,pharmaceutical composition or dietary supplement) is a composition thatcan be administered to a subject orally, e.g., a liquid composition suchas a solution. In some embodiments, the composition is a solidcomposition, for example, a lyophilite, which can be further processedprior to administering the composition to a subject, for example, thesolid composition can be further processed to form a liquid compositionsuch as a solution.

The pharmaceutical compositions of this invention may be administeredorally. Compositions suitable for oral administration may be in the formof capsules, cachets, pills, tablets, lozenges (using a flavored base,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a compound of the invention(s) as an activeingredient.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres.

In some embodiments, the compound of formula (V) is administered withone or more additional agents such as vitamins or other dietarysupplements.

Methods of Use

Folid acid and derivatives thereof can be found in found in naturallyfresh foods. In certain embodiments, a compound of formula (V) may serveas biological precursors to folic acid, e.g., a bioavailable precursorto folid acid. In certain other embodiments, a compound of formula (V)may be used as a dietary supplement. Compositions and oral dosage formsof the compound of formula (V) can be administered to a subject in needof folid acid, for example a pregnant female.

Methods of Making Compounds as Described Herein

The compounds of formula (I) and (V) described herein can be made usinga variety of synthetic techniques.

Scheme 1 above is an exemplary synthetic scheme that depicts arepresentative synthesis of compounds of formula (V) described herein.Calcium leucovorin 1 is reacted with formic acid to produce tricycle 2also referred to as ALV. Compounds of formula (V) can also be producedusing a variety of synthetic techniques.

Scheme 2 above is an exemplary synthetic scheme that depicts arepresentative synthesis of compounds of formula (I) described herein.ALV 2 is treated with sodium borohydride to produce compound 3.Compounds of formula (I) can also be produced using a variety ofacceptable synthetic techniques.

In embodiments, the methods described herein can be used to produce anenantiomerically enriched product.

Reaction Mixtures

The present invention also refers to a reaction mixture comprising acompound of formulas (II), (V) or (I), e.g., a reaction mixture that ispresent during a method or process described herein.

In certain embodiments, the methods or reaction mixtures describedherein further comprise a solvent. In certain embodiments, the solventis an aqueous solvent (e.g., water). In certain embodiments, the solventis an organic solvent. In certain embodiments, the solvent is an aproticsolvent. Exemplary organic solvents include, but are not limited to,benzene, toluene, xylenes, methanol, ethanol, isopropanol, acetonitrile,acetone, ethyl acetate, ethyl ether, tetrahydrofuran, methylenechloride, carbon tetrachloride, N-methylpyrrolidinone (NMP),N-methylmorpholine (NMM), dichloroethane and chloroform, or a mixturethereof.

In certain embodiments, the reaction is carried out below roomtemperature, e.g., a cooled reaction such as a reaction at a temperatureof 0° C. or lower. In certain embodiments, the reaction is carried outabove room temperature, e.g., by heating, e.g., from 25° C.-40° C. Incertain embodiments, the reaction occurs under an inert atmosphere, e.g,an atmosphere of an inert gas such as nitrogen or argon. In certainembodiments, the reaction takes place under anhydrous conditions, e.g.,conditions that are substantially free of water.

In some embodiments, the compounds described herein are in a reactionmixture comprising a solvent, e.g., as a mixture such as a solution or aheterogeneous mixture. The reaction mixture can be free of compoundsthat would react with or degrade a compound described herein e.g., thereaction mixture can be substantially free of water and/or substantiallyfree of any reactive gases.

Incorporation by Reference

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

EXAMPLES General Experimental Procedures Example 1: Preparation ofTricycle (4) from Calcium Leucovorin (6)

A 30 L jacketed reactor was fitted with a complete stir shaft assemblyand thermocouple. The reactor was charged with HCOOH (88%, 14.3 L),followed by conc HCl (2.5 L), and then water (1103 mL). Calciumleucovorin (1; 3942 g) was added to the reactor with stirring, followedby additional HCOOH (990 mL). After stirring for 17 h at ambienttemperature, crude 2 was collected by vacuum filtration, and washed withEtOH (3×8 L). The damp filter cake was dried under vacuum at 75° C. to aconstant weight. Yield: ˜2,740 g.

Example 2: Conversion of Anhydroleucovorin (2) to Calcium Salt of(6S)-methylfolate (3)

A 30 L jacketed reactor was fitted with a complete stir shaft assembly,thermocouple and heated/refrigerated recirculator. Water (15 L) andsodium hydroxide pellets (254 g) were added to the reactor and allowedto mix until dissolved. Sodium borohydride (1046 g) was added andallowed to mix until dissolved. The reactor was cooled to <8° C. andanhydroleucovorin (2, 3300 g) was slowly added over ˜1-3 hours. Thecontents of the reactor were allowed to mix for 1 hour at roomtemperature and then sodium borohydride (231 g) was slowly added. Thecontents of the reactor were allowed to mix for 1 hour at roomtemperature and then sodium borohydride (114 g) was very slowly added.The contents of the reactor were allowed to mix for one hour at roomtemperature. The borate salts byproducts were isolated by vacuumfiltration and washed with water (1.5 L). The reactor was rinsed withwater to remove residual salts. The filtered solution was added to thereactor and pH adjusted to 7.0-7.4 by slowly adding concentratedhydrochloric acid (37%, ˜330 mL). An aqueous solution of calciumchloride (19%, 5300 g) was slowly added over ˜10-30 minutes to effectprecipitation. The contents of the reactor were cooled to 0-8° C. andallowed to mix 12-16 hours. The crude 3 was collected by vacuumfiltration and washed with water (3 L) followed by methanol (3 L). Thedamp filter cake was added to the reactor, reslurried in methanol (30 L)for 2 hours at room temperature, filtered and washed with methanol (5L). The damp filter cake was added to the reactor, refluxed in methanol(30 L) for 2 hours, filtered warm and washed with methanol (5 L). Themethanol reflux procedure was repeated. The isolated solids were driedinvacuo at 60° C. to a constant weight. Yield ˜2,300 g.

What is claimed is:
 1. A method for making a compound of formula (V):

the method comprising cyclizing a compound of formula (II) to make acompound of formula (V):

wherein R¹ and R² are each independently selected from H, OH, NH₂, C₁₋₆alkyl or aryl; R³ is H, C₁₋₆ alkyl or aryl; Y is halo; and X is Na, K,Mg or Ca.
 2. The method of claim 1, wherein the cyclization step iscarried out in the presence of formic acid.
 3. The method of claim 2,further comprising a strong acid.
 4. The method of claim 3, wherein thestrong acid is HCl.
 5. The method of claim 2, further comprising asolvent.
 6. The method of claim 5, wherein the solvent is an aqueoussolvent.
 7. The method of claim 1, wherein R¹ is NH₂.
 8. The method ofclaim 1, wherein R² is OH.
 9. The method of claim 1, wherein R³ is H.10. The method of claim 1, wherein X is Ca.
 11. The method of claim 1,wherein Y is Cl.
 12. The method of claim 1, wherein R¹ is NH₂, R² is OH,R³ is H, X is Ca, and Y is Cl.
 13. The method of claim 1, the methodfurther comprising a reduction of a compound of formula (V) to make acompound of formula (I):